Preparation And Immune Effect Evaluation Of Grass Carp Reovirus Genotype Ⅱ Virus-like Particles

Grass carp haemorrhagic disease(GCHD)is a strong grass carp infectious disease caused by grass carp reovirus(GCRV),which has been listed as a second-class animal disease by the Ministry of Agriculture.GCRV is a member of the Aquareovirus genus in the family Reoviridae.GCRV is an icosahedral symmetric spherical particle with a diameter of 65~72 nm.It has a double-layer capsid and lacks of envelope membrane.The GCRV genome consists of 11 segmented double-stranded RNAs(dsRNA)and is divided into three genotypes: I,Ⅱ and ⅡI,according to the different gene sequences.Currently GCRV-Ⅱ is the main epidemic genotype.GCRV-Ⅱ is highly pathogenic and has a high mortality rate to grass carp.There is currently no effective drug treatment for the GCHD caused by GCRV-Ⅱ.Vaccine immunization is the most effective measure to prevent GCHD.Therefore,it is urgent to develop a safe and efficient GCRV-Ⅱ vaccine.Virus-like particles(VLPs)are highly structured hollow particles composed of one or more structural proteins of the virus,which are similar in shape to real virus particles.VLPs has some physical and chemical properties similar to natural virus,it can be taken up,processed and presented by antigen-presenting cells,and induce humoral and cellular immune responses,having the characteristics of high safety and good immunogenicity.Previous studies have shown that the structural proteins encoded by GCRV-Ⅱ S3,S6,S9 and S10 had good immunogenicity.In this study,we used the baculovirus expression system to construct GCRV-Ⅱ VLPs and selected GCRV-Ⅱ S3,S6,S9 and S10 genes to construct recombinant baculovirus.Multiple GCRV-Ⅱ VLPs were constructed through different combinations of recombinant baculovirus,and their immune effects were evaluated,which laid the foundation for the development of safe and effective GCRV-Ⅱ VLPs candidate vaccines.This research mainly includes the following four parts:1.Construction of GCRV-Ⅱ S3,S6,S9 and S10 gene recombinant baculovirus and expression of the target protein: In this study,four genes include of GCRV-Ⅱ S3,S6,S9 and S10 were selected to construct recombinant baculovirus pFBH-S3,pFBH-S6,pFBH-S9 and pFBH-S10,respectively.RT-PCR,indirect immunofluorescence(IFA)and Western blot were used to identify the construction and protein expression of recombinant baculovirus.The RT-PCR results showed that S3,S6,S9,and S10 mRNA were transcribed in Sf9 cells,but the IFA and Western blot results showed that only the proteins encoded by the S3,S6,and S10 genes were expressed,while the S9 gene was not,which indicated that recombinant baculoviruses pFBH-S3,pFBH-S6,and pFBH-S10 could successfully express the target proteins.2.GCRV-Ⅱ S6,S9 and S10 genes codon optimization and recombinant baculovirus construction and expression: The GENE PROJECT software was used to optimize the ORF codons of the S6,S9 and S10 genes with poor protein expression,then the recombinant baculovirus pFBH-opti-S6,pFBH-opti-S9 and pFBH-opti-S10 were constructed.The IFA and Western blot results showed that the proteins encoded by the S6,S9,and S10 genes,after codon optimization,were all expressed in higher levels compared to before optimization,indicating that the recombinant baculovirus pFBH-opti-S6,PFBH-opti-S9 and pFBH-opti-S10 could efficiently express the target protein.3.The construction of GCRV-Ⅱ VLPs based on six co-infection strategies with different combinations of recombinant baculovirus: The six recombinant baculoviruses were co-infected with different combinations to assemble VLPs,namely S3-S6-VLP,S3-S6-S10-VLPs and opti-S6-S9-S10-VLPs.The IFA results show that the proteins of interest were expressed in the three VLPs,respectively.Electron microscopic observation of ultra-thin cell slices and purified VLPs showed that the proteins expressed by different recombinant baculoviruses could all be self-assembled into VLPs in Sf9 insect cells,which are similar in shape to GCRV-Ⅱ and range in size from 40 nm to 60 nm.,Therefore,GCRV-Ⅱ S3-S6-VLPs,S3-S6-S10-VLPs and opti-S6-S9-S10-VLPs were successfully constructed in this study.4.Immune effects evaluation of three GCRV-Ⅱ VLPs: The grass carp was immunized with three purified VLPs,respectively.The experiment was set up with PBS control group,adjuvant groups and non-adjuvant groups.The results showed that three different VLPs all caused immune response in the fish after immunization.The results showed that three different VLPs could cause immune response in fish after immunization.ELISA results showed that the high OD450 values of grass carp serum were 0.84 和 0.83 in GCRV-Ⅱ S3-S6-S10-VLPs and opti-S6-S9-S10-VLPs with adjuvant immunization group.The relative quantitative PCR results showed that the mRNA relative expression levels of IFN,TLR-3,TLR-7,Mx and IgM gene in immunization groups were about 5 to 120 times higher than that in the PBS control group,which were significantly increased(P <0.05).The results of the challenge infection experiments showed that the relative protection rates of the groups of GCRV-Ⅱ S3-S6-VLP,S3-S6-S10-VLPs and opti-S6-S9-S10-VLPs without adjuvant were 41.67%,75.00%,58.33%.While the relative protection rates of the GCRV-Ⅱ S3-S6-VLP,S3-S6-S10-VLPs and opti-S6-S9-S10-VLPs groups with adjuvant were 58.33%,83.33%,and 79.17%.The above results showed that the three different VLPs of GCRV-Ⅱ had a certain protective effect on grass carp,of which S3-S6-S10-VLPs with adjuvant provided the best immune protection for the grass carp.In conclusion,this study used the Bac-to-Bac baculovirus expression system to constructed the GCRV-Ⅱ VLPs vaccine and evaluated its immune effect in this study.The results showed that the VLPs vaccine could induce grass carps to produce humoral and cellular immune responses,and had a certain immune protection effect on grass carps,of which the VLPs assembled by co-infected way with recombinant baculoviruses constructed from the three structural proteins S3,S6 and S10 have the best immune protection effect.This experiment not only provided technical reserves for immune prevention and control of grass carp haemorrhagic disease,but also provided an idea and method for the construction of virus-like particle vaccines for other aquatic animal diseases.